Isolation of 4-nerolidylcatechol from leaves of Piper peltatum L., and evaluation of larvicidal activity in mosquito vectors, with emphasis on Aedes aegypti (Diptera: Culicidae)

ABSTRACT

Synthetic chemical insecticides are widely used in population control of pests. Aedes aegypti is the primary vector of dengue, Zika, chikungunya and yellow fever to humans, and has proven resistance to chemical insecticides. As an alternative vector control method, the ethanolic extract of the leaves of Piper peltatum L. (Piperaceae) showed larvicidal activity against Ae. aegypti. Despite the wide medicinal use of this plant, the biological activity of its isolated constituents remains unexplored. In this sense, we isolated, identified and evaluated the larvicidal activity of 4-nerolidylcatechol (4-NC) from P. peltatum against Ae. aegypti, Culex quinquefasciatus and Anopheles darlingi, focusing on the larvicidal, adulticidal and genotoxic potential of 4-NC on Ae. aegypti. Larvae were captured in the city of Manaus, Amazonas state, Brazil. 4-NC was isolated from the extract of the leaves of P. peltatum via silica gel chromatography. This was identified using nuclear magnetic resonance spectroscopy and tested in Artemia franciscana (6.25, 12.5, 25, 50, 100 and 200 µg/mL). In the toxicity bioassay, Ae. aegypti larvae were exposed to 30, 50, 70, 90, and 110 µg/mL of 4-NC, while Cx. quinquefasciatus and An. darlingi were exposed to 6.25, 12.5, 25, 50 and 100 µg/mL. Ae. aegypti larvae were also subjected to 40 and 60 µg/mL of 4-NC (genotoxicity bioassay), and adult females to 62.5 to 1,000 µg/mL (adulticidal bioassay). The results of the 4-NC toxicity assays showed that there was 100% mortality in larvae of Ar. franciscana at the concentration of 200 µg/mL, with an LC₅₀ of 8.0 μg/mL. In the larvae of Ae. aegypti, mortality was 100%, with an LC₅₀ of 62 μg/mL. In larvae of Cx. quinquefasciatus, 97% mortality occurred, with an LC₅₀ of 52.3 μg/mL, and in An. darlingi larvae there was an 83% mortality rate, with an LC₅₀ of 55.8 μg/mL. In adults of Ae. aegypti, however, there was no adulticidal activity. In the larvae of Ae. aegypti, the genotoxic effect of 4-NC (40 and 60 µg/mL) showed significant frequency (p < 0.05) of cellular abnormalities (micronuclei, budding and nuclear bridges) of interphasic nuclei of neuroblasts and oocytes in relation to the negative control. This result may be associated with a decrease in oviposition of females, which was observed in two generations. We can confirm that 4-NC has larvicidal activity against Ae. aegypti, Cx. quinquefasciatus and An. darlingi. Although it does not present adulticidal activity in Ae. aegypti, it reduced the oviposition of females. Therefore, 4-NC seems to be a strong candidate for the development of an alternative method for the control of these mosquitoes in the immature phase.

Keywords:
Artemia franciscana; Bioassay; Dengue; Micronucleus; Toxicology

Introduction

Mosquitoes of the genera Aedes Meigen, 1818, Anopheles Meigen, 1818 and Culex Linnaeus, 1758, belong to the family Culicidae, and are distributed in two subfamilies – Anopheline and Culicinae. They are dipterans of epidemiological importance because some species transmit pathogens such as Plasmodium species, arboviruses, and microfilariae (Gaffigan et al., 2015; WHO, 2021).

Aedes (Stegomyia) aegypti (Linnaeus 1762) is the main vector of the four serotypes of dengue virus (DENV-1, -2, -3 and -4), chikungunya (CHIKV), yellow fever (YFV), and Zika (ZIKV) (Adler and Moncada-Álvarez, 2016). In Brazil, in 2021, 502,983 probable cases of dengue were reported, in addition to chikungunya and Zika, with 93,043 and 6,020 cases, respectively (Brasil, 2021a).

Malaria is an infectious disease caused by parasites of the genus Plasmodium that affected, in 2019, more than 241 million people worldwide (WHO, 2021). The mosquito Anopheles (Nyssorhynchus) darlingi Root, 1926 is considered the primary vector of malaria in South America (Tadei et al., 1988). In 2020, Brazil reported a total of 145,188 cases of malaria (Brasil, 2021b). Of this total, more than 99% occurred in the Brazilian Amazon region, and the main etiological agent was Plasmodium vivax (Brasil, 2021b). In this region, P. vivax infection predominates (83.7%) (Costa et al., 2012; Pimenta et al., 2015), while infections by Plasmodium falciparum (13.05%) and Plasmodium malariae (0.037%) are less common (Pimenta et al., 2015).

Culex quinquefasciatus Say (1823) is an anthropophilic mosquito that is distributed in several regions of the planet and presents epidemiological importance as a vector of several pathogens, such as Wuchereria bancrofti (Lai et al., 2000), West Nile virus, St. Louis, the Western Equine encephalitis virus (2012) and the Oropouche virus (Vasconcelos et al., 2011). The principal population control strategy of Ae. aegypti, An. darlingi and Cx. quinquefasciatus continues to be based on neurotoxic synthetic chemical insecticides from the group of organophosphates and pyrethroids (WHO, 2022). Although this type of intervention is crucial in endemic areas, continued use has contributed to the emergence and spread of insecticide resistance, and also affects non-target species and pollutes the environment (Valle et al., 2019, WHO, 2022). In this sense, health agencies need to discover new alternatives so as to reduce dependence on interventions that are based on synthetic chemical insecticides.

Plants are a source of promising secondary metabolites and have ovicidal, larvicidal, adulticidal and repellent activities against proven mosquito vectors (Ríos et al., 2017; Demirak & Canpolat, 2022). Essential oils (EOs) from Acacia nilotica and Eucalyptus globulus, for example, cause high mortality in larvae of Anopheles stephensi, Ae. aegypti and Cx. quinquefasciatus (Vivekanandhan et al., 2018a, 2020), and the extract of Acanthospermum hispidum showed larvicidal, pupicidal and adulticidal activity against these species (Vivekanandhan et al., 2018b). The EOs from Citrus aurantium and Cymbopogon citratus showed ovicidal activity against Ae. aegypti and Aedes albopictus (Moungthipmalai et al., 2023), and oil from Piper cordoncillo was toxic to Ae. aegypti larvae (Alonso-Hernández et al., 2023). The extract of Naringi crenulata was also toxic to Cx. quinquefasciatus (Pratheeba et al., 2019), and the combination of the nanoemulsion with essential oil from Eucalyptus grandis and Corymbia citriodora was highly toxic to larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus (Vivekanandhan et al., 2023).

Essential oils from plants of the genus Piper, family Piperaceae, have been extensively investigated for their insecticidal effect against mosquito vectors (França et al., 2021; Oliveira et al., 2022; Morais et al., 2023). Piper peltatum L., commonly known as cordoncillo, is a shrub that is easily found in edges of forests in the Amazon region (Guimarães et al., 2020). Although its methanolic extract has potent action against Ae. aegypti larvae (Mongelli et al., 2002), the effects of its chemical constituents against the species Anopheles and Culex were not found in the literature. Such activity of this substance may be related to 4-nerolidylcatechol (4-NC; C₂₁H₃₀O₂), which is one of the main components of P. peltatum (Pinto et al., 2006). It is a sesquiterpene that is isolated from the parts of this plant (roots and leaves) and makes up > 5% of the dry weight of its roots (Pinto et al., 2006). This substance has a number of proven biological activities, among them, antimalarial (Pinto et al., 2009; Bagatela et al., 2013), antitumor (Alves-Fernandes et al., 2020) and antioxidant (Lima et al., 2013).

Considering the importance of using Artemia salina Leach, 1812, or Artemia franciscana Kellogg, 1906, some studies correlate toxicity for Ar. salina as a biomarker with anticancer, antifungal and antimicrobial activities (Meyer et al., 1982). Both organisms are small in size, and easy to handle and acquire, thus allowing for rapid bioassays.

The micronucleus (MN) test has been used to detect clastogenic and aneugenic agents (Fenech et al., 2021). It has also been used in larvae of Ae. aegypti and Ae. albopictus, as the main genotoxic marker of natural and semisynthetic substances, such as dillapiole and its derivatives, demonstrating DNA damage (budding, polynucleated cells and interphasic and anaphasic bridges), according to Rafael et al. (2008) and Domingos et al. (2014). Studies with dillapiole and its semisynthetic derivatives from P. aduncum, with ovicidal, larvicidal, adulticidal and genotoxic effects, have generated promising results for the effective control of Ae. aegypti and Ae. albopictus (Rafael et al., 2008; Pinto et al., 2012; Domingos et al., 2014; Meireles et al., 2016; Santos et al., 2020; Silva et al., 2021).

Due to the absence of data on the toxic effect of 4-NC in mosquitoes, this research evaluated, for the first time, the toxic potential of this substance in Ae. aegypti (as a larvicide and adulticide) and in Cx. quinquefasciatus and An. darlingi (as a larvicide), using the microcrustacean Ar. franciscana as a marker of toxicity. In Ae. aegypti, the genotoxic aspects of 4-NC were also studied.

Materials and methods

Obtaining 4-nerolidylcatechol (4-NC), catechol and nerolidol

The isolation of the 4-nerolidylcatechol was made from the extract of the roots of P. peltatum, which was cultivated at Embrapa Amazônia Ocidental, Manaus, Amazonas state, Brazil (Pinto et al., 2006). The extraction was done from 150 g of dry roots subjected to ultrasound extraction (Ultrasonic Cleaner), using the mixture of CHCl₃:EtOH (1:1). The isolation of 4-NC was carried out from the extract by chromatography on silica gel (70-230 mesh, Merck), with the solvent mixture CHCl₃:EtOH (9: 1). The isolated 4-NC was identified by nuclear magnetic resonance spectrometry of hydrogen (1H), carbon (13C) and DEPT 135 (Varian, INOVA 500, CDCl3). Catechol and nerolidol were obtained from Sigma-Aldrich^® and used in this study to compare their larvicidal activity in Ar. franciscana.

Capture of mosquitoes

The capture of Ae. aegypti, and Cx. quinquefasciatus occurred in the Aleixo neighborhood (03º 05´ 29, 1´´ S, 59º 59´ 40, 7´´ W) and An. darlingi occurred in the Puraquequara lake (03º 06´ 18,89´´ S, 59º 84´ 36.92´´ W), in the municipality of Manaus, Amazonas state, Brazil. This activity was carried out with the authorization of the Chico Mendes Institute for Biodiversity Conservation (ICMBio), and Biodiversity Information and Authorization System (SISBIO), Brazil (permanent license number 32941, issued May 21^st, 2012 to Dr. Míriam Silva Rafael, INPA, Manaus, Amazonas state), for the collection of zoological material.

The specimens were transported to the insectarium at the Department of Society, Environment and Health, of the National Institute for Amazonian Research (COSAS/INPA), and were identified with the taxonomic identification keys of Forattini (2002) and Consoli and Lourenço-de-Oliveira (1994). The feeding of the larvae was with fishfood (TetraMin Tropical Flakes^®) and they were kept at 26± 2 ºC and 70± 5%, relative humidity. After the emergence of adult individuals, the feeding of mosquitoes was with glucose solution (5%) and the females with hamster blood (Mesocrisetus auratus), according to authorization No. 020/2017 from the Ethics Committee on the Use of Animals (CEUA) of the Central Vivarium at INPA.

Cytotoxicity test using Artemia franciscana

Cysts of Ar. franciscana Kellog were purchased from a commercial pet shop in Manaus, Amazonas, Brazil. The cysts were hatched in artificial seawater (2 gL^-1 NaHCO₃ + 8 gL^-1 NaCl) at room temperature (25 ºC) for 48 h, under constant illumination.

Third-instar larvae were exposed to 4-NC at concentrations of 6.25, 12.5, 25, 50, 100 and 200 µg/mL. The test took place in 24-well ELISA microplates, with the addition of 20 µL dimethylsufoxide (DMSO) for solubilization of each concentration of the substance, plus 1 mL of saline solution (35 g/L) for the 10 Ar. franciscana, in each replicate. For the negative control (NC), the addition of 20 µL of DMSO in saline solution occurred in 10 larvae. The bioassay was performed in triplicate and had its activity evaluated after 24 h of exposure in the absence of light.

The acquisition of the Ar. franciscana samples (1 vial of 10 g) was from PRODAC (aquarium products) International S.r.l, 35013, Cittadella PD Italy.

Larvicidal test of mosquitoes

A total of 1,300 Ae. aegypti, Cx. quinquefasciatus and An. darlingi larvae (3^rd and 4^th instar) were used in the 24-hour bioassay. These were distributed in five replicates, with ten individuals for each treatment (n = 5) and controls (n = 2), per species. Larvae of Ae. aegypti (n = 250) were exposed to concentrations 30, 50, 70, 90 and 110 µg/mL of 4-NC. Cx. quinquefasciatus (n = 250) and An. darlingi (n = 250) were submitted to concentrations of 6.25, 12.5, 25, 50 and 100 µg/mL. Each species tested had a negative control (NC; n = 50) in water and 0.02% DMSO (n = 150) and a positive control (PC; n = 50), which was temephos at 40 µg/mL (n = 150).

Aedes aegypti adulticide test

About 12 h before the adulticide test, also known as the biological bottle test (WHO 2006), with adaptations, 315 females Ae. aegypti were fed with blood. The solubilization process of the 4-NC was in acetone (w/v) in concentrations of 62.5 to 1,000 µg/mL. The NC was with acetone and the PC was with pyrethroid insecticide type II-alfacipermethrin. Then, Schott glass bottles (1 mL/vial) impregnated with different concentrations of 4-NC, NC with acetone and PC with alfacipermethrin received 15 previously bloodfed females each with blood, in triplicate. Readings took place every 15 min until 90 min had passed.

Aedes aegypti genotoxicity bioassay

First generation (G₁) third instar larvae of Ae. aegypti (n=600) were exposed to 4-NC at concentrations of 40 and 60 µg/mL (in triplicate/50 larvae each) as well as to the NC and the PC, for 4 h. Of these, 120 specimens had the brain ganglia (somatic cells) extracted for the cytological preparations, and the rest continued their development until adulthood. Then, the preparation of ovariole slides (gametic cells) of adult females (n=120) was performed, and the other samples were used to obtain G₂, according to the steps previously used in G₁.

Cytological preparations of Aedes aegypti

Cytological preparations of Ae. aegypti were performed according to Imai et al. (1988). For each concentration, brain ganglia of the G₁ and G₂ larvae (n=60) were hypotenized in buffer (0.8% sodium citrate in 0.005% colchicine) and incubated at 37 ºC, for 1 hour. Then, fixators I (ethanol: acetic acid: H₂0); II (ethanol: acetic acid) and III (glacial acetic acid) were added to the cell material, and dried at room temperature (RT). Giemsa solution in phosphate buffer 8%, pH 6.8, was used to stain the slides for 20 minutes. The manufacture of ovariole slides (n=30) of G₁ followed the same procedures used for the neuroblasts.

Analysis of slides and photographs

In each slide (n=10) 1,000 nuclei of each concentration of 4-NC and NC of G₁ and G₂ of neuroblasts, and of G₁ of oocytes were counted, totaling 90,000 cells. The abnormalities abnormal found in the cells were counted using a mechanical DigiTimer blood cell counter^ADAmTM-CelT (SATRA Technology Centre, Telford Way, Kettering, Northamptonshire, UK) and the microphotographs were obtained using an AxioCam MRcA camera under an Axioplan Zeiss light microscope (100× immersion objective with 1×, 1.25×, and 1.6× optovar; Carl Zeiss MicroImaging, Inc., Thornwood, NY, U.S.A.).

Oviposition of Aedes aegypti

The G₁ and G₂ generations of the adult female Ae. aegypti permitted the analysis in triplicate of the average oviposition of the individuals submitted to the concentrations of 40 and 60 µg/mL of 4-NC and the NC. The separation of adult females (n=10) aged between 3 and 5 days was in 50 mL paraffin cups, using screens, filter paper and 30 mL of drinking water.

Statistical analysis

Probit analysis determined the LC₅₀ and LC₉₀ and the percentage of mortality from the larvicidal test with Ar. franciscana, larvae and adult females of Ae. aegypti, and larvae of Cx. quinquefasciatus and An. darlingi. Using ANOVA (p<0.05), followed by the Tukey test (p <0.05), we observed the frequency of genotoxic damage of 4-NC in the neuroblasts and oocytes of Ae. aegypti. The mean oviposition of females between treatments and control, as well as between generations (G₁ and G₂) of this mosquito was significant.

Results

The isolation of 4-NC gave a yield of 8.6 g (44.1% of the total extracted) and a total yield of 5.7% based on the dried and ground root (m/m), and presented the molecular chemical structure C₂₁H₃₀O₂ (Fig. 1).

Figure 1
Molecular chemical structure of the substances 4-nerolidylcatechol (4-NC), isolated of Piper peltata, and catechol and nerolidol were obtained from Sigma-Aldrich^®, used in larvicidal and cytotoxicity bioassays.

4-NC was identified using ¹H and ¹³C NMR spectroscopy, and presented the following chemical displacements: NMR ¹H (CDCl₃; 500 MHz): 5.09 dd (J=10.8; 1.5 HZ); 5.02 dd (J= 17.4; 1.5 Hz); 5.99 dd (J= 17.4; 10.8 Hz); 1.95 m; 1.74 m; 5.07 m; 2.04 m; 1.81 m; 5.11 m; 6.75 dd (J= 8.4; 2.1 Hz); 6.80 d (J= 8.1 Hz); 6.85 d (J=2.0 Hz); 1.52 s; 1.60 s; 1.68 s; 1.33 s. NMR ¹³C (CDCl₃; 125 MHz): 140.9; 143.1; 114.9; 141.3; 119.1; 111.5; 147.0; 43.8; 41.1; 23.1; 124.3; 134.9; 39.7; 26.7; 124.5; 131.2; 25.7; 17.6; 15.9; 24.9. Calculated molecular weight: 314.4617 g/Mol. Density of 0.993 ± 0.06 g/cm³.

In Ar. franciscana, the substances 4-NC, catechol and nerolidol caused 100% mortality at the concentration of 200 μg/mL, and the LC₅₀ of the 4-NC was 8.0 μg/mL; catechol had an LC₅₀ of 23 μg/mL and nerolidol had an LC₅₀ of 9.4 μg/mL (Table 1).

Table 1
Lethal concentrations (LC₅₀ and LC₉₀) of 4-nerolidylcatechol, catechol and nerolidol in Artemia franciscana, and 4-nerolidylcatechol in larvae of Aedes aegypti, Culex quinquefasciatus and Anopheles darlingi, after exposure for 24 hours.

Organism	Lethal concentration	4-NC	Catechol	Nerolidol
Artemia franciscana *	(%)	100	100	100
	LC50 (µg/mL)	8.0 ± 1.0	23.0 ± 1.0	9.4 ± 1.0
		(7.8 – 9.6)	(19.6 – 27.1)	(8.4 – 10.6)
Aedes aegypti	(%)	100	0	13	100
	LC50 (µg/mL)	62 ± 0.8	-	-	33.8 ± 1.0
		(52.5 – 72.1)			(32.4 – 47.5)
	LC90 (µg/mL)	95 ± 2.0	-	-	103.2 ± 1.0
		(81.1 – 133.2)
Culex quinquefasciatus	(%)	97	100	100	100
	LC50 (µg/mL)	52.3 ± 1.0	209.5 ± 2.7	62.9 ± 0.6	-
		(46.1 – 59.3)	(190.3 – 222.5)	(51.5 – 69.1)
	LC90 (µg/mL)	99.2 ± 1.1	265.6 ± 2.0	125.7 ± 1.1
		(79.6 – 173.3)	(247.7 – 304.7)	(97.3 – 240.7)
Anopheles darlingi	(%)	83	-	-	100
	LC50 (µg/mL)	55.8 ± 1.0	-	-	-
		(46.6 – 66.9)
	LC90 (µg/mL)	139.3 ± 1.0	-	-	-
		(85.8 – 197.5)

• ^*
  C= 200 μg/mL

In larvae of Ae. aegypti, after 24 h, the 4-NC had caused 100% mortality at the concentration of 110 μg/mL and presented an LC₅₀ of 62 μg/mL and an LC₉₀ of 95 μg/mL. In the tests with the larvae of Cx. quinquefasciatus, the observed mortality was 97% at the concentration of 100 μg/mL, with an LC₅₀ of 52.3 μg/mL and an LC₉₀ of 99.2 μg/mL. In the larvae of An. darlingi, there was a mortality rate of 83% at the concentration of 100 μg/mL with an LC₅₀ of 55.8 μg/mL and an LC₉₀ of 139.3 μg/mL. For the catechol and nerolidol standards, 100% mortality was only achieved in Cx. quinquefasciatus with an LC₅₀ of 209.5 and an LC₉₀ of 265.6 μg/mL for catechol, and an LC₅₀ 62.9 and an LC₉₀ 125.7 μg/mL for nerolidol (Table 1).

The tested concentrations of 4-NC did not cause mortality Ae. aegypti in adulthood during the evaluation time of the test (90 min). This was perhaps due to this substance not being volatile and since it presents a high molecular weight.

The genotoxicity of substance 4-NC in neuroblasts and oocytes of Ae. aegypti, at the concentrations of 40 and 60 µg/mL showed the presence of nuclear and chromosomal alterations, such as micronucleus (MN), nuclear sprouts (NS), chromosomal breaks (CB), among other malformations, as shown in Fig. 2.

Figure 2
Microphotographs of abnormalities in interphasic and metaphasic nuclei of neuroblasts and oocytes of Aedes aegypti, stained with Giemsa (pH 5.8) and lacto-acetic orcein (2%). Arrows indicate: a – normal interphasic nuclei of neuroblasts of the NC group, G₁; b and c – micronuclei in interphasic nuclei of neuroblasts of G₂ (40 and 60 µg/mL), respectively; d – budding and telophasic bridging nucleus of neuroblasts (60 µg/mL, G₂); e – budding in interphasic nucleus of oocytes (40 µg/mL, G₁); f – normal metaphasic chromosomes (NC, G₁); g and h – chromosomal metaphases of neuroblasts showing achromatic secondary constriction (60 µg/mL, G₂) . Magnification: 1600×. Scale bar: 5 and 10 μm.

The frequency of nuclear and chromosomal damage in interphasic nuclei of neuroblasts of Ae. aegypti exposed to 4-NC (40 and 60 µg/mL) for 4 hours was significantly higher (p>0.05) when compared to the NC, but not between their treatments. The frequency of damage in individuals exposed to the substance also showed a significant increase in G₂ when compared with G₁ (Fig. 3A). In interphasic nuclei of oocytes of first-generation females exposed to 4-NC (40 and 60 µg/mL), there was a significant difference (p < 0.05) in the number of nuclear and chromosomal abnormalities in relation to the NC, but not between treatments (Fig. 3b).

Figure 3
Frequency of anomalies in interphase nuclei. a – damage to Aedes aegypti neuroblasts exposed to 4-NC for two generations. b – data on Ae. aegypti oocytes exposed to 4-NC in G₁.

Table 2 shows the decrease in the amount of eggs laid by female Ae. aegypti treated with 4-NC compared to untreated mosquitoes (control). The largest reduction in the number of eggs was with 40 µg/mL, in which the average number of eggs was 70.0 ± 5.1 in G₁, decreasing to 51.8 ± 6.0 in G₂; while in the control the average was 90.0 ± 4.6 and 82.0 ± 2.7 in G₁ and G₂, respectively.

Table 2
Mean and standard deviation of oviposition of adult females of Aedes aegypti survivors of the genotoxicity bioassay at concentrations 40 and 60 µg/mL of 4-neroldycatechol (4-NC), and negative control, for two generations (G₁ and G₂).

Compound	Concentration (µg/mL)	Generation
		G1	G2
Negative control	0	90.0 ± 4.6	82.0 ± 2.7
4-NC	40	70.0 ± 5.1	51.8 ± 6.0
	60	56.0 ± 3.2	41.5 ± 8.1

Discussion

In this study, the 4-NC used in the highest concentrations was cytotoxic in Ar. franciscana, and caused 100% mortality of the exposed individuals. These data corroborate those of Mongelli et al. (1999) who observed toxicity of the methanolic extract of P. peltata leaves (LC₅₀ 89 µg/mL) against Artemia salina.

In mosquitoes, the larvicidal activity of isolated natural substances is poorly reported in the literature. In the present study, the high larval mortality recorded in Ae. aegypti corroborates the work of Mongelli et al. (2002). 4-NC demonstrated high lethality in larvae of Ae. aegypti (LC₅₀ of 9.1 µg/mL and LC₅₀ of 38.7 µg/mL), respectively, and it is present in the methanolic extract of the leaves of P. peltata and in the chloroform/ethanolic extract of the roots of that plant, respectively in these studies. Other studies with extracts, essential oils and their derivatives have reported ovicidal, larvicidal, and adulticidal activities in Ae. aegypti (Rafael et al., 2008; Pinto et al., 2012; Ríos et al., 2017; França et al., 2021; Oliveira et al., 2022; Morais et al., 2023).

Rafael et al. (2008) used dillapiole and observed larval mortality of 3^rd instar Ae. aegypti at concentrations of 200 and 400 µg/mL (53% and 67%), respectively, after 72 hours. After 24 hours, derivatives of dillapiole ethyl ether (80 µg/mL) and n-butyl ether (30 µg/mL) killed 83.75 and 100% of the Ae. aegypti larvae, respectively (Domingos et al., 2014). The dillapiole methyl ether (DME), at a concentration of 140 µg/mL, made 97% of the eggs unviable and caused 99% larval mortality (Silva et al., 2019). Nerolidol, at concentrations of 100 and 200 μg/mL, also caused 100% mortality of the larvae (Meireles et al., 2021). Despite the high mortality caused by 4-NC in Ae. aegypti larvae, there was no adulticidal activity, probably because it is not a very volatile substance and has a high molecular weight. Studies are lacking to document this toxic activity in mosquitoes; however, Pinto et al. (2012) tested dillapiole, isodillapiole and butyl, ethyl and propyl ethers derived from dillapiole isolated from Piper aduncum at a concentration of 100 µg/mL in Ae. aegypti, and recorded 100% mortality in adults.

On the other hand, genotoxicity studies in Ae. aegypti neuroblasts recorded frequent budding and micronuclei in interphasic nuclei in larvae exposed to 4-NC for 4 h, which was significant (p < 0.05) in relation to the NC, and chromosomal breaks in metaphases. It is likely that the action of genotoxic agents on DNA occurs during de-espiralization, a moment of greater vulnerability of the molecule (Rafael et al., 2008; Fenech et al., 2021). This can lead to the formation of micronuclei and damage to the mitotic spindle mechanism involved in chromosome separation (Albertini et al., 2000), while breaks in chromosomal arms, also observed in Ae. aegypti, are associated with the regions of secondary constrictions on chromosome 3 of this mosquito (Rafael et al., 2008). Studies by Valadares et al. (2007) evaluated the mutagenicity and antimutagenicity of 4-NC in bone marrow cells of mice, and noted that this substance has no mutagenic effect on these cells and that there was a protective effect against genotoxicity. Barros et al. (2005) evaluated the mutagenicity of the ethanolic extract of 4-NC in mice and observed the absence of mutagenic activity. On the other hand, Alves-Fernandes et al. (2020) noted low genotoxicity of 4-NC in in vitro test in human cells. Dillapiole and its derivatives, even at low concentrations, showed toxic effects against eggs, larvae and adults of Ae. aegypti and Ae. albopictus (Pinto et al., 2012), and was able to alter genes that are important to the development of individuals (Meireles et al., 2016; Silva et al., 2021).

In this study, in adult females previously treated in the larval phase with 4-NC, there was a slight decline in fertility. This result is consistent with the data in Ae. aegypti (Domingos et al., 2014) and Ae. albopictus treated with dillapiole ethyl ether and dillapiole n-butyl ether (Meireles et al., 2016). These authors associated the reduced oviposition rate of adult females with DNA damage, as observed in Ae. aegypti after treatment with E-isodillapiole (Santos et al., 2020) and dillapiole methyl ether, and that even the smallest concentrations can cause damage to the genome of individuals and interfere with their function (Silva et al., 2021).

The data reported, in addition to the findings of the present study, reinforce the importance of toxicity analyses of candidate substances for effective vector control of Ae. aegypti, Cx. quinquefasciatus and An. darlingi, and genotoxicity in Ae. aegypti, because of the resistance of these mosquitoes to synthetic insecticides. In this sense, dillapiole n-butyl ether was cytotoxic for Ae. aegypti and Ae. albopictus (Domingos et al., 2014; Meireles et al., 2016). This substance, with LD_{25, 50} and ₈₀, however, was promising in hepatic, renal and cardiac tissues of Balb/C mice, since it was not cytotoxic at the lowest concentration (Viana Cruz et al., 2020) in relation to those previously tested in Ae. aegypti and Ae. albopictus. This suggests future testing of the toxicity and genotoxicity of 4-NC against immature forms of Ae. aegypti in field environments, and other non-target insects and mammals, for clarification of its larvicidal potential.

Conclusions

The toxicity of the substance 4-NC showed high mortality rates in eggs and of 3^rd instar Ae. aegypti larvae, in Cx. quinquefasciatus and An. darlingi larvae and in Ar. franciscana, but was absent in adult subjects of Ae. aegypti. Genotoxicity, in the Ae. aegypti bioassay with 4-NC showed nuclear and chromosomal damage and reduced oviposition of adult females. These data suggest the need for future research on the application and effects of 4-NC as an alternative and effective tool in the control of Ae. aegypti in the field, and its application in other organisms.

Acknowledgments

We are grateful to Dr. Adalberto Luis Val for financial support through project INCT ADAPTA II/INPA/CNPq/FAPEAM (process No. 465540/2014-7), and to FAPEAM/SEPLANCTI/Governo do Estado do Amazonas, POSGRAD, project No. 002/2016, and FAPEAM/SEDECTI/Governo do Estado do Amazonas - Edital POSGRAD/FAPEAM 2019.

References

• Adler, P. H., Moncada-Álvarez, L. I., 2016. Medical entomology, a necessity. Rev. Salud Publica (Bogota) 18, 163-164. http://doi.org/10.15446/rsap.v18n2.57077
  » http://doi.org/10.15446/rsap.v18n2.57077
• Albertini, R. J., Anderson, D., Douglas, G. R., Hagmar, L., Hemminki, K., Merlo, F., Natarajan, A. T., Norppa, H., Shuker, D. E. G., Tice, R., Waters, M. D., Aitio, A., 2000. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutat. Res. Rev. Mutat. Res. 463 (2), 111-172. http://doi.org/10.1016/S1383-5742(00)00049-1
  » http://doi.org/10.1016/S1383-5742(00)00049-1
• Alonso-Hernández, N., Granados-Echegoyen, C., Vera-Reyes, I., Pérez-Pacheco, R., Arroyo-Balán, F., Valdez-Calderón, A., Espinosa-Roa, A., Loeza-Concha, H. J., Villanueva-Sánchez, E., García-Pérez, F., Diego-Nava, F., 2023. Assessing the Larvicidal Properties of Endemic Campeche, Mexico Plant Piper cordoncillo var. apazoteanum (Piperaceae) against Aedes aegypti (Diptera: Culicidae) Mosquitoes. Insects 14 (4), 312. http://doi.org/10.3390/insects14040312
  » http://doi.org/10.3390/insects14040312
• Alves-Fernandes, D. K., Oliveira, É. A., Hastreiter, A. A., Faião-Flores, F., Felipe-Silva, A. S., Turato, W., Fock, R. A., Maria-Engler, S. S., Barros, S. B. D. M., 2020. In vivo antitumoral effect of 4-nerolidylcatechol (4-NC) in NRAS-mutant human melanoma. Food Chem. Toxicol. 141, 111371. http://doi.org/10.1016/j.fct.2020.111371
  » http://doi.org/10.1016/j.fct.2020.111371
• Bagatela, B. S., Lopes, A. P., Fonseca, F. L. A., Andreo, M. A., Nanayakkara, D. N. P., Bastos, J. K., Perazzo, F. F., 2013. Evaluation of antimicrobial and antimalarial activities of crude extract, fractions and 4-nerolidylcathecol from the aerial parts of Piper umbellata L. (Piperaceae). Nat. Prod. Res. 27 (23), 2202-2209. http://doi.org/10.1080/14786419.2013.821123
  » http://doi.org/10.1080/14786419.2013.821123
• Barros, S., Ropke, C. D., Sawada, T. C. H., Silva, V. V., Pereira, S. M. M., Barros, S. B. M., 2005. Assessment of acute and subchronic oral toxicity of ethanolic extract of Pothomorphe umbellata L. Miq (Pariparoba). RBCF Rev. Bras. Cienc. Farm. 41 (1), 53-61. http://doi.org/10.1590/S1516-93322005000100005
  » http://doi.org/10.1590/S1516-93322005000100005
• Brasil, 2021a. Boletim Epidemiológico: monitoramento dos casos de arboviroses urbanas causados por vírus transmitidos pelo mosquito Aedes (dengue, chikungunya e zika), semanas epidemiológicas 1 a 47, 2021. Available in: https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/boletins/epidemiologicos/edicoes/2021/boletim_epidemiologico_svs_44-2.pdf (accessed 30 April 2022).
  » https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/boletins/epidemiologicos/edicoes/2021/boletim_epidemiologico_svs_44-2.pdf
• Brasil, 2021b. Boletim Epidemiológico: malária 2021. Available in: https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/boletins/boletins-epidemiologicos/especiais/2021/boletim_epidemiologico_especial_malaria_2021.pdf (accessed 25 April 2022).
  » https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/boletins/boletins-epidemiologicos/especiais/2021/boletim_epidemiologico_especial_malaria_2021.pdf
• Consoli, R.A.G.B., Lourenço-de-Oliveira, R., 1994. Principais mosquitos de importância sanitária no Brasil, Editora Fiocruz, Rio de Janeirol, 228 p. http://doi.org/10.7476/9788575412909
  » http://doi.org/10.7476/9788575412909
• Costa, F. T. M., Lopes, S. C. P., Albrecht, L., Ataíde, R., Siqueira, A. M., Souza, R. M., Russell, B., Renia, L., Marinho, C. R. F., Lacerda, M. V. G., 2012. On the pathogenesis of Plasmodium vivax malaria: perspectives from the Brazilian field. Int. J. Parasitol. 42 (12), 1099-1105. http://doi.org/10.1016/j.ijpara.2012.08.007
  » http://doi.org/10.1016/j.ijpara.2012.08.007
• Demirak, S., Canpolat, E., 2022. Plant-based bioinsecticides for mosquito control: impact on insecticide resistance and disease transmission. Insects 13 (2), 162-162. http://doi.org/10.3390/insects13020162
  » http://doi.org/10.3390/insects13020162
• Domingos, P. R. C., Pinto, A. C. S., Santos, J. M. M., Rafael, M. S., 2014. Insecticidal and genotoxic potential of two semi-synthetic derivatives of dillapiole for the control of Aedes (Stegomyia) aegypti (Diptera: culicidae). Mutat. Res. Genet. Toxicol. Environ. Mutagen. 772, 42-54. http://doi.org/10.1016/j.mrgentox.2014.07.008
  » http://doi.org/10.1016/j.mrgentox.2014.07.008
• Fenech, M., Knasmueller, S., Knudsen, L. E., Kirsch-Volders, M., Deo, P., Franzke, B., Stopper, H., Andreassi, M., Bolognesi, C., Dhillon, V. S., Laffon, B., Wagner, K., Bonassi, S., 2021. “Micronuclei and Disease” special issue: aims, scope, and synthesis of outcomes. Mutat. Res. Rev. Mutat. Res. 788, 108384. http://doi.org/10.1016/j.mrrev.2021.108384
  » http://doi.org/10.1016/j.mrrev.2021.108384
• Forattini, O.P., 2002. Culicidologia médica: identificação, biologia e epidemiologia, Vol. 2. EDUSP, São Paulo.
• França, L. P., Amaral, A. C. F., Ferreira, J. L. P., Ramos, A. S., Chaves, F. C., Tadei, W. P., Silva, J. R. A., 2021. Piper capitarianum essential oil: a promising insecticidal agent for the management of Aedes aegypti and Aedes albopictus. Environ. Sci. Pollut. Res. Int. 28 (8), 9760-9776. http://doi.org/10.1007/s11356-020-11148-6
  » http://doi.org/10.1007/s11356-020-11148-6
• Gaffigan, T.V., Wilkerson, R.C., Pecor, J.E., Stoffer, J.A., Anderson, T., 2015. Systematic Catalog of Culicidae - Walter Reed Biosystematics. Available in: https://bugguide.net/node/view/878135 (accessed 25 April 2022).
  » https://bugguide.net/node/view/878135
• Guimarães, E. F., Medeiros, E. V. S. S., Queiroz, G. A., 2020. Piper In: Jardim Botânico do Rio de Janeiro – JBRJ (Ed.), Flora do Brasil 2020. Rio de Janeiro: JBRJ. Available in: https://floradobrasil2020.jbrj.gov.br/FB20317 (accessed 30 February 2022).
  » https://floradobrasil2020.jbrj.gov.br/FB20317
• Imai, H. T., Taylor, R. W., Crosland, M. W. J., Crozier, R. H., 1988. Modes of spontaneous chromosomal mutation and karyotype evolution in ants with reference to the minimum interaction hypothesis. Jpn. J. Genet. 63 (2), 159-185. http://doi.org/10.1266/jjg.63.159
  » http://doi.org/10.1266/jjg.63.159
• Lai, C. H., Tung, K. C., Ooi, H. K., Wang, J. S., 2000. Competence of Aedes albopictus and Culex quinquefasciatus as vector of Dirofilaria immitis after blood meal with different microfilarial density. Vet. Parasitol. 90 (3), 231-237. http://doi.org/10.1016/S0304-4017(00)00242-9
  » http://doi.org/10.1016/S0304-4017(00)00242-9
• Lima, E., Pinto, A. C. S., Nogueira, K., Silva, L., Almeida, P., Vasconcellos, M., Chaves, F., Tadei, W. P., Pohlit, A., 2013. Stability and antioxidant activity of semi-synthetic derivatives of 4-Nerolidylcatechol. Molecules 18 (1), 178-189. http://doi.org/10.3390/molecules18010178
  » http://doi.org/10.3390/molecules18010178
• Meireles, S. F., Domingos, P. R. C., Pinto, A. C. S., Rafael, M. S., 2016. Toxic effect and genotoxicity of the semisynthetic derivatives dillapiole ethyl ether and dillapiole n -butyl ether for control of Aedes albopictus (Diptera: culicidae). Mutat. Res. Genet. Toxicol. Environ. Mutagen. 807, 1-7. http://doi.org/10.1016/j.mrgentox.2016.07.003
  » http://doi.org/10.1016/j.mrgentox.2016.07.003
• Meireles, S.F., Pinto, A.C.S., Silva, J.S., Nascimento-Neto, J.F., Cruz, D. L. V., Rafael, M.S., 2021. Atividade larvicida da substância nerolidol contra Aedes aegypti (Diptera: Culicidae). In: Silva, J.S., Lima, R.C., Soares, S.C., Batista, J.S., Formiga, K.M. (Eds.), DNAlogando sobre conservação e biologia evolutiva na Amazônia. Editora Itacaiúnas, Ananideua, pp. 33-38. http://doi.org/10.36599/itac-dna.0006
  » http://doi.org/10.36599/itac-dna.0006
• Meyer, B., Ferrigni, N., Putnam, J., Jacobsen, L., Nichols, D., McLaughlin, J., 1982. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 45 (5), 31-34. http://doi.org/10.1055/s-2007-971236
  » http://doi.org/10.1055/s-2007-971236
• Moungthipmalai, T., Puwanard, C., Aungtikun, J., Sittichok, S., Soonwera, M., 2023. Ovicidal toxicity of plant essential oils and their major constituents against two mosquito vectors and their non-target aquatic predators. Sci. Rep. 13 (1), 2119. http://doi.org/10.1038/s41598-023-29421-2
  » http://doi.org/10.1038/s41598-023-29421-2
• Mongelli, E., Romano, A., Desmarchelier, C., Coussio, J., Ciccia, G., 1999. Cytotoxic 4-Nerolidylcatechol from Pothomorphe peltate Inhibits Topoisomerase I Activity. Planta Med. 65 (4), 376-378. http://doi.org/10.1055/s-2006-960793
  » http://doi.org/10.1055/s-2006-960793
• Mongelli, E., Coussio, J., Ciccia, G., 2002. Investigation of the larvicidal activity of Pothomorphe peltata and isolation of the active constituent. Phytother. Res. 16 (S1), 71-72. http://doi.org/10.1002/ptr.753
  » http://doi.org/10.1002/ptr.753
• Morais, L. S., Sousa, J. P. B., Aguiar, C. M., Gomes, C. M., Emarque, D. P., Albernaz, L. C., Espindola, L. S., 2023. Edible plant extracts against Aedes aegypti and validation of a Piper nigrum L. ethanolic extract as a natural insecticide. Molecules 28 (3), 1264-1264. http://doi.org/10.3390/molecules28031264
  » http://doi.org/10.3390/molecules28031264
• Oliveira, A. C., Simões, R. C., Lima, C. A. P., Silva, F. M. A., Nunomura, S. M., Roque, R. A., Tadei, W. P., Nunomura, R. C. S., 2022. Essential oil of Piper purusanum C.DC (Piperaceae) and its main sesquiterpenes: biodefensives against malaria and dengue vectors, without lethal effect on non-target aquatic fauna. Environ. Sci. Pollut. Res. Int. 29 (31), 47242-47253. http://doi.org/10.1007/s11356-022-19196-w
  » http://doi.org/10.1007/s11356-022-19196-w
• Pinto, A.C.S., Pena, E.A., Pohlit, A.M., Chaves, F.C.M., 2006. Biomass production in cultivated Pothomorphe peltata Miq. (Piperaceae) as a function of harvest time in Manaus, Amazonas State, Brazil. Rev. Bras. Plantas Med. 8, 98-101.
• Pinto, A. C. S., Silva, L. F. R., Cavalcanti, B. C., Melo, M. R. S., Chaves, F. C. M., Lotufo, L. V. C., Moraes, M. O., Andrade-Neto, V. F., Tadei, W. P., Pessoa, C. O., 2009. New antimalarial and cytotoxic 4-nerolidylcatechol derivatives. Eur. J. Med. Chem. 44 (6), 2731-2735. http://doi.org/10.1016/j.ejmech.2008.10.025
  » http://doi.org/10.1016/j.ejmech.2008.10.025
• Pinto, A. C. S., Nogueira, K. L., Chaves, F. C. M., Silva, L. V. S., Tadei, W. P., Pohlit, A. M., 2012. Adulticidal activity of dillapiol and semi-synthetic derivatives of dillapiol against adults of Aedes aegypti (L.) (Culicidae). J. Mosq. Res. 2, 1-7. http://doi.org/10.5376/jmr.2012.01.0001
  » http://doi.org/10.5376/jmr.2012.01.0001
• Pimenta, P. F., Orfano, A. S., Bahia, A. C., Duarte, A. P. M., Ríos-Velásquez, C. M., Melo, F. F., Pessoa, F. A. C., Oliveira, G. A., Campos, K. M. M., Villegas, L. M., Rodrigues, N. B., Nacif-Pimenta, R., Simões, R. C., Monteiro, W. M., Amino, R., Traub-Cseko, Y. M., Lima, J. B. P., Barbosa, M. G. V., Lacerda, M. V. G., Tadei, W. P., Secundino, N. F. C., 2015. An overview of malaria transmission from the perspective of Amazon Anopheles vectors. Mem. Inst. Oswaldo Cruz 110 (1), 23-47. http://doi.org/10.1590/0074-02760140266
  » http://doi.org/10.1590/0074-02760140266
• Pratheeba, T., Vivekanandhan, P. A. K., Nur Faeza, A. K., Natarajan, D., 2019. Chemical constituents and larvicidal efficacy of Naringi crenulata (Rutaceae) plant extracts and bioassay guided fractions against Culex quinquefasciatus mosquito (Diptera: culicidae). Biocatal. Agric. Biotechnol. 19, 101137-101137. http://doi.org/10.1016/j.bcab.2019.101137
  » http://doi.org/10.1016/j.bcab.2019.101137
• Rafael, M. S., Hereira-Rojas, W. J., Roper, J. J., Nunomura, S. S., Tadei, W. P., 2008. Potential control of Aedes aegypti (Diptera: Culicidae) with Piper aduncum L. (Piperaceae) extracts demonstrated by chromosomal biomarkers and toxic effects on interphase nuclei. Genet. Mol. Res. 7 (3), 772-781. http://doi.org/10.4238/vol7-3gmr481
  » http://doi.org/10.4238/vol7-3gmr481
• Ríos, N., Stashenko, E. E., Duque, J. E., 2017. Evaluation of the insecticidal activity of essential oils and their mixtures against Aedes aegypti (Diptera: culicidae). Rev. Bras. Entomol. 61 (4), 307-311. http://doi.org/10.1016/j.rbe.2017.08.005
  » http://doi.org/10.1016/j.rbe.2017.08.005
• Santos, L. H. F., Domingos, P. R. C., Meireles, S. F., Bridi, L. C., Pinto, A. C. S., Rafael, M. S., 2020. Genotoxic effects of semi-synthetic isodillapiole on oviposition in Aedes aegypti (Linnaeus, 1762) (Diptera: culicidae). Rev. Soc. Bras. Med. Trop. 53, e20200467. http://doi.org/10.1590/0037-8682-0467-2020
  » http://doi.org/10.1590/0037-8682-0467-2020
• Silva, J.S., Pinto, A.C.S., Santos, L.H.F., Rafael, M.S., 2019. Efeito ovicida e larvicida do éter metil dilapiol (EMD) em Aedes aegypti, Manaus-AM. In: Salgado, Y.C.S. (Ed.), Patologias: doenças parasitárias. Athena, Ponta Grossa, pp. 192-204. Available in: https://www.atenaeditora.com.br/catalogo/post/efeito-ovicida-e-larvicida-do-eter-metil-dilapiol-emd-em-aedes-aegypti-manaus-am (accessed 25 April 2022). http://doi.org/10.22533/at.ed.97819180323
  » https://www.atenaeditora.com.br/catalogo/post/efeito-ovicida-e-larvicida-do-eter-metil-dilapiol-emd-em-aedes-aegypti-manaus-am» http://doi.org/10.22533/at.ed.97819180323
• Silva, J. S., Pinto, A. C. S., Santos, L. H. F., Silva, L. J. S., Cruz, D. L. V., Rafael, M. S., 2021. Genotoxic and mutagenic effects of methyl ether dillapiole on the development of Aedes aegypti (Diptera: culicidae). Med. Vet. Entomol. 35 (4), 556-566. http://doi.org/10.1111/mve.12533
  » http://doi.org/10.1111/mve.12533
• Tadei, W. P., Thatcher, B. D., Santos, J. M. M., Scarpassa, V. M., Rodrigues, I. B., Rafael, M. S., 1988. Ecologic observations on Anopheline vectors of malaria in the Brazilian Amazon. Am. J. Trop. Med. Hyg. 59 (2), 325-335. http://doi.org/10.4269/ajtmh.1998.59.325
  » http://doi.org/10.4269/ajtmh.1998.59.325
• Valadares, M. C., Castro, N. C., Cunha, L. C., 2007. Synadenium umbellatum: citotoxicidade e danos ao DNA de células da medula óssea de camundongos. RBCF Rev. Bras. Cienc. Farm. 43 (4), 631-638. http://doi.org/10.1590/S1516-93322007000400017
  » http://doi.org/10.1590/S1516-93322007000400017
• Valle, D., Bellinato, D. F., Viana-Medeiros, P. F., Lima, J. B. P., Martins Junior, A. J., 2019. Resistance to temephos and deltamethrin in Aedes aegypti from Brazil between 1985 and 2017. Mem. Inst. Oswaldo Cruz 114, e180544. http://doi.org/10.1590/0074-02760180544
  » http://doi.org/10.1590/0074-02760180544
• Vasconcelos, H. B., Nunes, M. R. T., Casseb, L. M. N., Carvalho, V. L., Pinto da Silva, E. V., Silva, M., Casseb, S. M. M., Vasconcelos, P. F. C., 2011. Molecular epidemiology of oropouche virus, Brazil. Emerg. Infect. Dis. 17 (5), 800-806. http://doi.org/10.3201/eid1705.101333
  » http://doi.org/10.3201/eid1705.101333
• Vivekanandhan, P., Venkatesan, R., Ramkumar, G., Karthi, S., Senthil-Nathan, S., Shivakumar, M. S., 2018a. Comparative analysis of major mosquito vectors response to seed-derived essential oil and seed pod-derived extract from Acacia nilotica. Int. J. Environ. Res. Public Health 15 (2), 388. http://doi.org/10.3390/ijerph15020388
  » http://doi.org/10.3390/ijerph15020388
• Viana Cruz, D. L., Sumita, T. C., Silva, L. F. M., Silva, J. S., Pinto, A. C. S., Barcellos, J. F. M., Rafael, M. S., 2020. Histopathological, cytotoxicological, and genotoxic effects of the semi-synthetic compound dillapiole n-butyl ether in Balb/C mice. J. Toxicol. Environ. Health A 83 (17-18), 604-615. http://doi.org/10.1080/15287394.2020.1804026
  » http://doi.org/10.1080/15287394.2020.1804026
• Vivekanandhan, P., Senthil‐Nathan, S., Shivakumar, M. S., 2018b. Larvicidal, pupicidal and adult smoke toxic effects of Acanthospermum hispidum (DC) leaf crude extracts against mosquito vectors. Physiol. Mol. Plant Pathol. 101, 156-162. http://doi.org/10.1016/j.pmpp.2017.05.005
  » http://doi.org/10.1016/j.pmpp.2017.05.005
• Vivekanandhan, P., Usha-Raja-Nanthini, A., Valli, G., Subramanian, S. M., 2020. Comparative efficacy of Eucalyptus globulus (Labill) hydrodistilled essential oil and temephos as mosquito larvicide. Nat. Prod. Res. 34 (18), 2626-2629. http://doi.org/10.1080/14786419.2018.1547290
  » http://doi.org/10.1080/14786419.2018.1547290
• Vivekanandhan, P., Panikar, S., Veeran, S., Usha-Raja-Nanthini, A., Shivakumar, M. S., 2023. Toxic and synergetic effect of plant essential oils along with nano-emulsion for control of three mosquito species. J. Nat. Pestic. Res. 5, 100045-100045. http://doi.org/10.1016/j.napere.2023.100045
  » http://doi.org/10.1016/j.napere.2023.100045
• World Health Organization – WHO, 2006. Guidelines for Testing Mosquito Adulticides for Indoor Residual Spraying and Treatment of Mosquito Nets. WHO, Geneva. Available in: https://iris.who.int/handle/10665/69296 (accessed 25 April 2022).
  » https://iris.who.int/handle/10665/69296
• World Health Organization – WHO, 2021. World Malaria Report 2021. WHO, Geneva.
• World Health Organization – WHO, 2022. Manual for Monitoring Insecticide Resistance in Mosquito Vectors and Selecting Appropriate Interventions. WHO, Geneva. Available in: https://www.who.int/publications/i/item/9789240051089 (accessed 25 April 2022).
  » https://www.who.int/publications/i/item/9789240051089

Edited by

Associate Editor: Maria Sallum

Publication Dates

• Publication in this collection
  14 June 2024
• Date of issue
  2024

History

• Received
  24 Aug 2023
• Accepted
  09 Feb 2024